1. Field of the Invention
This invention relates to a cell death accelerator for inducing cell death and a cell death inhibitor for inhibiting cell death.
2. Description of the Related Art
For the purpose of culturing cells in vitro, serum is generally added to a synthetic culture medium in a concentration range of about 10-20%. The medium is a pH-balanced salt solution containing various nutrients such as vitamins, amino acids, and sugars. When serum or plasma is highly concentrated in the medium, beyond this range however, cell death is induced.
The cell death-inducing activity is seen in all the sera or plasma so far tested regardless of source (species or age), or heat treatment to inactivate complement. Two types of cell death are now generally recognized. The first is passive, resulting from lack of oxygen, mechanical crush injury, or other extreme change in the extracellular environment. The second type of cell death is called apoptosis, an active energy-requiring intracellular process that culminates in DNA fragmentation and osmotic lysis of the cell. The latter is an autonomous physiological death sometimes referred to as programmed cell death, and it occurs normally during development of the nervous system, skin, and other epithelial organs. Morphologically, apoptosis is characterized by blebbing of the plasma membrane and nuclear condensation. These visible changes are accompanied by synthesis of a specific protein(s) including nuclease followed by fragmentation of chromosomal DNA. Several apoptosis-related genes have already been identified in the nematode C. elegans and in mammalian lymphocytes.
Apoptosis is now the focus of much attention, because it appears to be crucial for normal tissue and organogenesis, neural development, and immune reactions to foreign antigens including those borne by infectious viruses.
The serum contains some components, i.e., proteins such as albumin and globulin, salts such as NaCl, KCl and CaCl.sub.2, vitamins, more than twenty amino acids, such as glycine, cystine, cysteine, alanine and tryptophan, and other components. Heretofore adequate investigation of the individual functions of each component in the serum has not been carried out. The present inventors disclosed that amino acids, such as cystine and cysteine, when added to the serum, decrease the toxic effect of the serum and accelerates growth and/or multiplication of cultured cells in Japanese Patent Application No. 270719/1990 filed on Oct. 9, 1990. At present, however, investigation of the toxic factor itself has not been carried out at all, and the characterization of components which can inhibit the toxicity has been insufficient. In the noted Japanese Application, the inventors disclosed that cysteine and cystine promoted cell growth and/or multiplication, and that tryptophan can prevent cell death.
Recently, Evan et al. (1992) reported that cultured fibroblasts died when the mitotic cycle was interrupted during c-myc expression. Serum-induced cell death may involve a similar mechanism. Cells entering the mitotic cycle in response to an excess of growth signals from serum may die after interruption of the cycle by thiol deficiency. Previously, we reported that a cell-death-inducing activity was present in a low molecular weight fraction of serum (M.W.&lt;1,000) (Kurita and Namiki, 1993a). Subsequently, it was found that this activity was inhibited by thiols. It was water-soluble, heat-resistant, and had charcoal affinity. Approximate molecular weight of the factor was approximately 100-200 dalton upon size-sieve HPLC. In addition to low molecular weight factors, traces of macromolecules in the fraction are now thought to be necessary for cell death. The latter may act as a death signal, and c-myc may be also involved in the signal transduction. Further studies are needed to address these issues. Nevertheless, serum-induced cell death appears to be a type of apoptosis resulting from a disturbance in thiol metabolism.